Accel-NGS® 2S PCR-Free DNA Library Kit

PCR-free NGS Prep with Low Input Capability for Illumina® Platforms

Eliminate Sequencing Bias from Polymerase with PCR-Free Preps

The Accel-NGS 2S PCR-Free DNA Library Kit, designed for Illumina platforms, enables preparation for high complexity NGS libraries from double-stranded DNA. PCR-free libraries can be made with unbiased coverage from cfDNA inputs as low as 10 ng and sheared DNA as low as 100 ng. The Accel-NGS 2S PCR-Free Kit offers greater than 90% conversion efficiency for applications pertaining to whole genome sequencing of relatively less damaged samples such as liquid biopsy and cfDNA. This technology delivers the following features and benefits:

Using four incubations, this protocol repairs both 5’ and 3’ termini and sequentially attaches Illumina adapter sequences to the ends of fragmented dsDNA.

Bead-based clean-ups are used to remove oligonucleotides and small fragments, and to change enzymatic buffer composition between steps. Different bead-to-sample ratios are utilized for different input quantities and insert sizes.

For PCR-free applications, the resulting functional library is ready for quantification and sequencing on the Illumina platform. Alternatively, an optional PCR step may be used to increase yield of indexed libraries, which then may be quantified and sequenced.

Generate Higher Complexity Libraries than the Leading Competitive Kit

Swift Biosciences technology yields more unique library molecules than the competitive kit which allows for greater coverage of the genome in each sequencing run.

Libraries were made PCR-free from HapMap DNA NA12878 (Coriell) and sequenced on the HiSeq. Competitor I data is from public access files. Estimated library size was calculated by Picard MarkDuplicates (picard.sourceforge.net).

Cover Extreme Base Composition Regions Better

Libraries were made PCR-free from HapMap DNA NA12878 (Coriell) and sequenced on the HiSeq. Competitor I data is from public access files.

Expect Even Coverage with Human DNA Sequencing

Accel-NGS 2S PCR-free offers outstanding evenness of coverage for large, complex genomes such as human. This holds true for both PCR-free and libraries requiring PCR amplification. This enables high quality libraries to be constructed from various inputs of human DNA without compromising the quality of the data.

Accel-NGS 2S libraries were constructed from Coriell HapMap NA12878 DNA, 100 ng PCR-free or 10 ng with 6 cycles of PCR. Sequencing was performed on an Illumina HiSeq 2500 in rapid run mode. Data was analyzed using BWA (Li and Durbin, 2010) and Picard CollectGcBiasMetrics (picard.sourceforge.net). The coverage vs. theoretical plot on the left panel demonstrates that libraries constructed from both 10 ng input with 6 cycles of PCR and 100 ng PCR-free show a comparable distribution to the theoretical. The right panel demonstrates that a library constructed from 100 ng PCR-free with Accel-NGS 2S provides even coverage across a range of GC content.

Validation on the Illumina MiSeq

The data below demonstrate the performance of the Accel-NGS 2S PCR-Free DNA Library Kit with microbial genomes of varying base composition that were prepared and sequenced on the Illumina MiSeq by a third party.

Yes, we recommend using a qPCR-based assay to quantify starting material with amplicons that are sized to indicate the amplifiable content of the sample. There are several commercially-available qPCR-based input quantification kits available.

The sequences of the adapters in Accel-NGS 2S libraries are identical to Illumina TruSeq® LT adapters (Single Indexing) or Illumina TruSeq HT adapters (Dual Indexing), but are constructed in a proprietary manner. The adapters and indices are supplied directly from Swift Biosciences in a 2S Indexing Kit.

The Accel-NGS 2S kits have been validated with 165 bp (cfDNA), 200 bp (gDNA), 350 bp (gDNA), and 450 bp (gDNA) fragments. If working with fragments of another size, please contact Tech Support for recommendations.

Accel-NGS 2S Kits construct high complexity libraries from dsDNA input through two dedicated repair steps and sequential ligation of adapters. Repair I dephosphorylates 5’ termini of input dsDNA to prevent chimera formation. Repair II performs 3’ end repair and polishing. Ligation I performs 3’ ligation of the P7 adapter, and Ligation II performs 5’ ligation of the P5 adapter. These separate, sequential ligation steps prevent formation of adapter dimers and enable independent optimization of each adapter attachment to each terminus. Following Ligation II, an Optional PCR Step can be performed to amplify the library, if necessary.

Repair I dephosphorylates 5’ termini of input dsDNA to prevent chimera formation. Chimeric library molecules can affect alignment metrics, as they are composed of fragments that will be read as a single library molecule, but will align to different genomic locations. Repair II performs 3’ end repair and polishing, which is critical to efficient ligation of adapters.

Yes, for customers that have not yet prepared the Ligation I master mix, we recommend pausing the library preparation following the Post-Repair II Step. We recommend re-suspending the SPRI beads in 10 µl of Low EDTA TE following the Post-Repair II SPRI Step, and storing the samples at 4°C. When you are ready to resume the library preparation, adjust the Ligation I master mix to include only 10 µl of Low EDTA TE rather than 20 µl. This will account for the 10 µl of Low EDTA TE that the beads have been stored in, so that the final volume of the Ligation I reaction remains the same. Proceed to run the samples with the Ligation I Thermocycler Program.

For customers that have already prepared the Ligation I master mix who desire a safe stopping point prior to the Post-Ligation II SPRI Step, please contact Tech Support for recommendations.

Following the standard SPRI Cleanup steps that are specified in the protocol will result in left side size selection (removal of small DNA only). For customers with samples that contain large fragments (>600 bp), we recommend removal of these prior to library quantification as these large library molecules will contribute to the library concentration, but will not cluster well on the flow cell. For information on performing a right side size selection (removal of large DNA only) or double size selection (removal of both small and large DNA), please see the SPRIselect User Guide published by Beckman Coulter.

For high quality, fragmented gDNA, the Accel-NGS 2S Kits exhibit a conversion efficiency of 20-70%.* For circulating, cell-free DNA (cfDNA), higher conversion efficiencies are observed (up to 90%). This increase can be attributed to the relatively undamaged ends of cfDNA resulting from enzymatic cleavage in the blood and the narrow size distribution of cfDNA. For low quality DNA samples, such as those from FFPE, conversion rates can be lower due to unrepairable damage on the ends of DNA.

Lower than expected yields can usually be attributed to inaccurate quantification of input DNA or inefficient recovery of DNA during the bead-based clean-up steps. While NanoDrop® or Qubit® may be acceptable for high quality DNA samples, quantification by a qPCR-based method is recommended to ensure accuracy of input DNA.

For damaged DNA samples, please be aware that small fragments (< 100 bp) will be excluded by the standard SPRI bead ratios indicated in the protocol.

There are two common causes for abnormal migration of Accel-NGS 2S library molecules.

The first cause applies to PCR-free libraries only. The secondary structure of sequencing adapters in PCR-free Accel-NGS 2S libraries results in abnormal migration and overestimation of library size. On the Agilent High Sensitivity Chip, it is normal and expected to observe 200 bp insert PCR-free libraries to migrate to a ~500 bp peak, and for 350 bp insert PCR-free libraries to migrate to a ~800 bp peak. Performing a few cycles of PCR on the library resolves the adapter secondary structure and results in library molecules that migrate true to size.

The second cause applies to libraries that have been over-amplified. Too many cycles of PCR can deplete primer concentration in the reaction, resulting in the formation of library molecule heteroduplex structures that will migrate abnormally. Denaturation of these heteroduplex structures – with a denaturing gel, for example – will result in library molecules that migrate true to size. Over-amplified libraries can still be sequenced, as they will be denatured just prior to loading on the flow cell, but customers should aim to perform the minimum number of PCR cycles necessary to avoid undesirable PCR duplicates.

Yes, Accel-NGS 2S kits are readily compatible with automation instruments. Scripts are in the process of being written for the Beckman Coulter Biomek® FXP and NXP, the PerkinElmer SciClone® and SciClone Janus®, the TECAN Fluent™, the Hamilton Microlab® STAR™, and the Eppendorf epMotion®.